The expression and potential function of bone morphogenetic proteins 2 and 4 in bovine trophectoderm

<p>Abstract</p> <p>Background</p> <p>Bone morphogenetic proteins (BMPs) were first described for their roles in bone formation, but they now also are known to possess additional activities, including those relating to embryogenesis. The objectives of this work were to 1) determine if peri-attachment bovine conceptuses and bovine trophoblast cells (CT1) contain transcripts for <it>BMP2 </it>and <it>4</it>, an innate inhibitor noggin (<it>NOG</it>), and BMP2/4 receptors (<it>BMPRII</it>, <it>ACVR1</it>, <it>BMPR1A</it>, <it>BMPR1B</it>), and 2) determine if BMP2 or 4 supplementation to CT1 cells affects cell proliferation, differentiation or trophoblast-specific gene expression.</p> <p>Methods</p> <p>RNA was isolated from day 17 bovine conceptuses and CT1 cells. After RT-PCR, amplified products were cloned and sequenced. In other studies CT1 cells were treated with BMP2 or 4 at various concentrations and effects on cell viability, cell differentiation and abundance of IFNT and CSH1 mRNA were evaluated.</p> <p>Results</p> <p>Transcripts for <it>BMP2 </it>and <it>4 </it>were detected in bovine conceptuses and CT1 cells. Also, transcripts for each BMP receptor were detected in conceptuses and CT1 cells. Transcripts for <it>NOG </it>were detected in conceptuses but not CT1 cells. Cell proliferation was reduced by BMP4 but not BMP2 supplementation. Both factors reduced <it>IFNT </it>mRNA abundance but had no effect on <it>CSH1 </it>mRNA abundance in CT1 cells.</p> <p>Conclusions</p> <p>The BMP2/4 ligand and receptor system presides within bovine trophectoderm prior to uterine attachment. BMP4 negatively impacts CT1 cell growth and both BMPs affect IFNT mRNA abundance.</p

Autoimmunization of Ewes Against Pregnancy-associated Glycoproteins Does Not Interfere with the Establishment and Maintenance of Pregnancy

doi:10.1017/S1751731109004145Pregnancy-associated glycoproteins (PAGs) are a large grouping of placental proteins that belong to the aspartic peptidase gene family. Although useful to detect pregnancy in ruminant species, the function of these molecules is unclear. Several PAGs expressed by trophoblast binucleate cells can enter the maternal circulation, suggesting that they could have a systemic role in altering maternal physiology. The objective of this work was to examine whether these circulating placental antigens were important in pregnancy by actively immunizing ewes against them. PAGs were purified by pepstatin-affinity chromatography and conjugated to the immunogenic protein, keyhole limpet hemocyanin (KLH). Ewes were immunized with PAG-KLH conjugate (n522) or with KLH alone (n59), and bred to intact rams. Blood samples, collected on Day 0 (day of estrus), Day 10, Days 15 to 25 and weekly throughout pregnancy, were analyzed for PAG by an ELISA. On Day 30, pregnancy was confirmed by ultrasound. Ewes immunized against PAG-KLH produced a range of reactive anti-PAG titers, whereas all immunized ewes had high anti-KLH immunoreactivity. PAGs became detectable in the anti-KLH (control) ewes at Day 21.662.2 of pregnancy. Those ewes immunized against PAGs (n57), that had very low immunoreactivity toward PAGs, had measurable PAG by Day 22.961.3, and their PAG serum profiles throughout pregnancy did not differ from the controls. Those exhibiting moderate to high anti-PAG immunoreactivity (n515), had significantly lower PAG concentrations than controls, with antigen not becoming detectable until Day 48.1615.6. The decrease in circulating PAG in the immunized animals did not correlate with changes in pregnancy rates, lamb number or lamb birth weight. These results suggest that while PAGs may play a role in maintaining pregnancy, their major contribution is likely to be at the fetal-maternal interface. Their actions at extra-placental sites are presumably of more secondary importance.This work was supported by NIH Grant HD21896 and the Animal Reproductive Biology Group of the University of Missouri Food for the 21st Century Program

Bovine Blastocyst-Derived Trophectoderm and Endoderm Cell Cultures: Interferon Tau and Transferrin Expression as Respective In Vitro Markers

Continuous cultures of bovine trophectoderm (CT-1 and CT- 5) and bovine endoderm (CE-1 and CE-2) were initiated and maintained on STO feeder cells. CT-1 and CT-5 were derived from the culture of intact, 10- to 11-day in vitro-produced blastocysts. CE-1 and CE-2 were derived from the culture of immunodissected inner cell masses of 7- to 8-day in vitro-produced blastocysts. The cultures were routinely passaged by physical dissociation. Although morphologically distinct, the trophectoderm and endoderm both grew as cell sheets of polarized epithelium (dome formations) composed of approximately cuboidal cells. Both cell types, particularly the endoderm, grew on top of the feeder cells for the most part. Trophectoderm cultures grew faster, relative to endoderm, in large, rapidly extending colonies of initially flat cells with little or no visible lipid. The endoderm, in contrast, grew more slowly as tightly knit colonies with numerous lipid vacuoles in the cells at the colony centers. Ultrastructure analysis revealed that both cell types were connected by desmosomes and tight junctional areas, although these were more extensive in the trophectoderm. Endoderm was particularly rich in rough endoplasmic reticulum and Golgi apparatus indicative of cells engaged in high protein production and secretion. Interferon tau expression was specific to trophectoderm cultures, as demonstrated by reverse transcription-polymerase chain reaction, Western blot, and antiviral activity; and this property may act as a marker for this cell type. Serum protein production specific to endoderm cultures was demonstrated by Western blot; this attribute may be a useful marker for this cell type. This simple coculture method for the in vitro propagation of bovine trophectoderm and endoderm provides a system for assessing their biology in vitro

Exposure to maternal obesity alters gene expression in the preimplantation ovine conceptus

Abstract Background Embryonic and fetal exposure to maternal obesity causes several maladaptive morphological and epigenetic changes in exposed offspring. The timing of these events is unclear, but changes can be observed even after a short exposure to maternal obesity around the time of conception. The hypothesis of this work is that maternal obesity influences the ovine preimplantation conceptus early in pregnancy, and this exposure will affect gene expression in embryonic and extraembryonic tissues. Results Obese and lean ewe groups were established by overfeeding or normal feeding, respectively. Ewes were then bred to genetically similar rams. Conceptuses were collected at day 14 of gestation. Morphological assessments were made, conceptuses were sexed by genomic PCR analysis, and samples underwent RNA-sequencing analysis. While no obvious morphological differences existed between conceptuses, differentially expressed genes (≥ 2-fold; ≥ 0.2 RPKM; ≤ 0.05 FDR) were detected based on maternal obesity exposure (n = 21). Also, differential effects of maternal obesity were noted on each conceptus sex (n = 347). A large portion of differentially expressed genes were associated with embryogenesis and placental development. Conclusions Findings reveal that the preimplantation ovine conceptus genome responds to maternal obesity in a sex-dependent manner. The sexual dimorphism in response to the maternal environment coupled with changes in placental gene expression may explain aberrations in phenotype observed in offspring derived from obese females